CAJ | 학술논문

目的建立一种高度敏感和特异的人隐孢子虫的RT-PCR-ELISA检测方法.方法根据人隐孢子虫与其他隐孢子虫粘附蛋白p23基因的多重比对,设计一对引物(其中p1引物带有生物素标记)用来扩增含高变区目的片段;采用RT-PCR方法扩增目的片段,扩增产物与探针引物进行液相杂交,将杂交产物与微孔板上包被的链霉亲和素结合,再与辣根过氧化酶标记的抗地高辛抗体反应.用所建立的方法对22份临床样本进行检测,并与常规检测方法进行比较.结果所建立的RT-PCR-ELISA检测方法具有高特异性和敏感性,比常规的PCR方法灵敏度提高了100(0.08:8 ng)左右.临床检测结果显示,该方法的检出率高达86%(19/2),而RT-PCR、蔗糖漂浮法和抗酸染色法的检出率仅为27%(6/22)、27%(6/22)和50%(11/22).结论本试验成功建立了人隐孢子虫的RT-PCR-ELISA检测方法,检出率比常规检测方法高.
목적 건립일충고도민감화특이적인은포자충적RT-PCR-ELISA검측방법.방법 근거인은포자충여기타은포자충점부단백p23기인적다중비대,설계일대인물(기중p1인물대유생물소표기)용래확증함고변구목적편단;채용RT-PCR방법확증목적편단,확증산물여탐침인물진행액상잡교,장잡교산물여미공판상포피적련매친화소결합,재여랄근과양화매표기적항지고신항체반응.용소건립적방법대22빈림상양본진행검측,병여상규검측방법진행비교.결과 소건립적RT-PCR-ELISA검측방법구유고특이성화민감성,비상규적PCR방법령민도제고료100(0.08:8 ng)좌우.림상검측결과현시,해방법적검출솔고체86%(19/2),이RT-PCR、자당표부법화항산염색법적검출솔부위27%(6/22)、27%(6/22)화50%(11/22).결론 본시험성공건립료인은포자충적RT-PCR-ELISA검측방법,검출솔비상규검측방법고.
To establish a highly sensitive and specific method to detect the presence of Cryptosporidium homini, the RT-PCR-ELISA assay was tried, in which the primer with a biotin-labeled probe was designed to amplify fragment containing the highly variable region by multiple alignment between p23 gene of C.hominis and other Cryptosporidium spp. The RT-PCR was used to amplify the target fragment, and the amplified product was used to hybridize with the probe primer. The hybridized product was then captured on micro-plate wells coated with streptavidin and reacted with anti-digoxin antibody labeled with horse-radish peroxidase. This method of testing was then used for the detection of C.hominis in 22 clinical specimens and compared with the conventional methods of testing. It was demonstrated that the RT-PCR--ELISA for the detection of C.hominis was proved to be quite sensitive and specific. Its sensitivity was 100 times higher than that of the general PCR. From the result of clinic detection, the detection rate of RT-PCR-ELISA assay attained to 86%(19/22), while those of RT-PCR, sucrose floating method and anti-acid staining were 27%, 27% and 50% respectively. This result indicates that the RT-PCR-ELISA assay is more sensitive to detect C.hominis than the other three methods of testing.